PROJECT SUMMARY/ABSTRACT - PROJECT 3 Tim-3 modulates immunity to the important human pathogens HIV, HCV, and Mycobacterium tuberculosis (Mtb). The interaction between Tim-3 and its ligands can induce T cell exhaustion: a state characterized by progressive loss of effector functions leading to impairment of T cell mediated immunity. Importantly, reversing T cell exhaustion is a potential therapeutic strategy for the treatment of cancer and chronic viral infection. We have previously shown that the Tim3/Gal9 interaction activates macrophages to suppress intracellular Mtb growth and our new data shows that Tim-3 promotes IFN? production by T cells. We detect two distinct Tim-3+ T cell subsets in the lungs of infected mice: 1) T cells that express Tim-3 but not other exhaustion markers (which we refer to as Tim3+Exh-); and 2) T cells that express Tim3 in combination with other exhaustion markers (Tim3+Exh+). The former subset, which has not been described during chronic viral infection or cancer, behaves as effector T cells in mice with TB. The latter subset appears to be exhausted. Experiments using blocking antibodies or knockout mice to interrupt Tim-3 signaling improves Mtb disease outcome, which suggests that these interventions lead to improved immunity. Thus, two (opposing) paradigms exist for how Tim-3 signaling affects microbial immunity: (1) Tim-3 expression promotes exhaustion of pathogen-specific T cells during chronic infections and cancers; and (2) Tim-3 binding to Gal9 promotes T cell and macrophage antimicrobial activity during acute infection. Our observation that both effector and exhausted T cells express Tim-3 leads us to hypothesize that the the capacity of Tim-3 to positively or negatively regulate immunity is influenced by (a) the cytokines IL-27 and IL-21, which affect its expression; (b) its ligands, Gal-9 and the recently identified CEACAM-1; and (c) other exhaustion molecules that are co- expressed with Tim-3 is co-expressed. We recently identified CEACAM-1 as a second Tim-3 ligand and showed that the trans-interaction between Tim-3 and CEACAM-1 is required to suppress effector T cell function and to maintain T cell tolerance/exhaustion. We will test the hypothesis that the interaction between Tim-3 and its two ligands Gal-9 and CEACAM-1, elicit distinct T cell outcomes. The chronic TB model is perfect for these experiments. Not only is T cell dysfunction believed to play a major role in the pathogenesis of TB, but also we made the novel observation that two distinct Tim3-expressing T cell subsets [Tim3+Exh- and Tim3+Exh+] differ in functions [effector vs. exhausted, respectively]. Therefore, TB provides an important opportunity to identify the mechanism of how Tim-3 ligand selection and the effect of other regulatory signals (e.g., cytokines) affect T cell effector function and dysfunction. The overarching goal of this project is to understand how these factors regulate the dual functions of Tim-3 to modulate its net beneficial or detrimental effect on microbial immunity during chronic infection.